Endocrine-disrupting chemicals (EDCs) are widespread environmental contaminants that affect a wide range of endocrine and neurobiological functions, including onset of puberty, reproductive function, and display of affective behaviors. Laboratory animal models provide direct evidence for such EDC actions, and epidemiological data in humans support correlations between EDC exposures and reproductive and neurobehavioral deficits. While exposure to EDCs likely occurs throughout the lifetime, this proposal seeks to investigate the effects of EDC exposure during two specific critical periods of development: late gestation and puberty. Gestation is the major life stage during which actions of gonadal steroid hormones organize the brain to cause sexually dimorphic structural and functional changes in the brain and behavior. Likewise, pubertal increases in release of gonadal steroid hormones act upon these pathways both to continue these organizational processes, as well as to activate those pathways organized earlier in life. This novel proposal will identify interactions between these EDC exposures periods on behavioral and molecular endpoints using a two-hit model, testing the hypothesis that earlier exposure may amplify effects of pubertal exposure. Here, I will utilize a well-established EDC model of polychlorinated biphenyls, using Aroclor 1221 (A1221), a lightly chlorinated mixture. Previous work has shown that gestational exposure to A1221 perturbs neurobiological development, and has latent effects on neuroendocrine functions, gene expression in the brain, and behavior. For my two-hit model, rats will receive exposures to A1221 or vehicle during gestation and early adolescence. This results in four groups (first hit prenatal, second hit adolescent): A1221-A1221; Veh-Veh; A1221-Veh; Veh-A1221. A primary outcome will be the display of social and anxiety-related behaviors outcomes during both adolescence and adulthood, as these behaviors are hormone-sensitive and associated with sex-biased mental health disorders in humans. Additionally, I will quantify corresponding changes in gene expression of selected neuropeptides in brain regions previously shown as important in the display of these behaviors. Finally, because at least part of the mechanism for brain organization is through actions of hormones on DNA methylation to program how these genes function later in life, the research will also determine if molecular epigenetic changes are a mechanism for long-term changes in gene expression in response to EDC exposure during critical developmental periods. These goals meet the NIEHS strategic goal to investigate the effects of the environment on genome structure and function, including epigenetic regulation of biological processes, and to identify critical windows of susceptibility to the effects of environmental exposures. Moreover, the proposal extends our understanding of environmental influences on behavioral and molecular endpoints relevant to reproductive and mental health disorders in humans.